title: Sulfation of the bikunin chondroitin sulfate chain determines heavy chain·hyaluronan complex formation.
abstract: Inter-α-trypsin inhibitor (IαI) is a complex comprising two heavy chains (HCs) that are covalently bound by an ester bond to chondroitin sulfate (CS), which itself is attached to Ser-10 of bikunin. IαI is essential for the trans-esterification of HCs onto hyaluronan (HA). This process is important for the stabilization of HA-rich matrices during ovulation and some inflammatory processes. Bikunin has been isolated previously by anion exchange chromatography with a salt gradient up to 0.5 M NaCl and found to contain unsulfated and 4-sulfated CS disaccharides. In this study, bikunin-containing fractions in plasma and urine were separated by anion exchange chromatography with a salt gradient of 0.1-1.0 M NaCl, and fractions were analyzed for their reactivity with the 4-sulfated CS linkage region antibody (2B6). The fractions that reacted with the 2B6 antibody (0.5-0.8 M NaCl) were found to predominantly contain sulfated CS disaccharides, including disulfated disaccharides, whereas the fractions that did not react with this antibody (0.1-0.5 M NaCl) contained unsulfated and 4-sulfated CS disaccharides. IαI in the 0.5-0.8 M NaCl plasma fraction was able to promote the trans-esterification of HCs to HA in the presence of TSG-6, whereas the 0.1-0.5 M NaCl fraction had a much reduced ability to transfer HC proteins to HA, suggesting that the CS containing 4-sulfated linkage region structures and disulfated disaccharides are involved in the HC transfer. Furthermore, these data highlight that the structure of the CS attached to bikunin is important for the transfer of HC onto HA and emphasize a specific role of CS chain sulfation.
authors.affiliation: Graduate School of Biomedical Engineering, The University of New South Wales, Sydney, New South Wales 2052, Australia. m.lord@unsw.edu.au
authors.forename: Megan S
authors.initials: MS
authors.lastname: Lord
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Day
authors.forename: Peter
authors.initials: P
authors.lastname: Youssef
authors.forename: Lisheng
authors.initials: L
authors.lastname: Zhuo
authors.forename: Hideto
authors.initials: H
authors.lastname: Watanabe
authors.forename: Bruce
authors.initials: B
authors.lastname: Caterson
authors.forename: John M
authors.initials: JM
authors.lastname: Whitelock
mesh_terms: D000510:Alpha-Globulins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D002236:Carbohydrate Conformation; D002809:Chondroitin Sulfates / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D005109:Extracellular Matrix / Q000737:chemistry / Q000378:metabolism; D005260:Female; D006801:Humans; D006820:Hyaluronic Acid / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008297:Male; D010060:Ovulation / Q000502:physiology
chemical_list: D000510:Alpha-Globulins; C016001:alpha-1-microglobulin; D006820:Hyaluronic Acid; D002809:Chondroitin Sulfates
keywords: Bikunin; Chondroitin Sulfate; Extracellular Matrix Proteins; Glycosaminoglycan; Hyaluronan; Inter-α-trypsin Inhibitor; Proteoglycan; Proteoglycan Structure